Participating as the Cornell-Gdansk group, we have used our physics-based coarse-grained UNited RESidue (UNRES) force field to predict protein structure in the 11th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP11). Our methodology involved extensive multiplexed replica exchange simulations of the target proteins with a recently improved UNRES force field to provide better reproductions of the local structures of polypeptide chains. All simulations were started from fully extended polypeptide chains, and no external information was included in the simulation process except for weak restraints on secondary structure to enable us to finish each prediction within the allowed 3-week time window. Because of simplified UNRES representation of polypeptide chains, use of enhanced sampling methods, code optimization and parallelization and sufficient computational resources, we were able to treat, for the first time, all 55 human prediction targets with sizes from 44 to 595 amino acid residues, the average size being 251 residues. Complete structures of six single-domain proteins were predicted accurately, with the highest accuracy being attained for the T0769, for which the CαRMSD was 3.8 Å for 97 residues of the experimental structure. Correct structures were also predicted for 13 domains of multi-domain proteins with accuracy comparable to that of the best template-based modeling methods. With further improvements of the UNRES force field that are now underway, our physics-based coarse-grained approach to protein-structure prediction will eventually reach global prediction capacity and, consequently, reliability in simulating protein structure and dynamics that are important in biochemical processes. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://www.unres.pl/ CONTACT: [email protected].

UNLABELLED: The differences between observed and predicted (13)C(α) chemical shifts can be used as a sensitive probe with which to detect possible local flaws in protein structures. For this reason, we previously introduced CheShift, a Web server for protein structure validation. Now, we present CheShift-2 in which a graphical user interface is implemented to render such local flaws easily visible. A series of applications to 15 ensembles of conformations illustrate the ability of CheShift-2 to locate the main structural flaws rapidly and accurately on a per-residue basis. Since accuracy plays a central role in CheShift predictions, the treatment of histidine (His) is investigated here by exploring which form of His should be used in CheShift-2. AVAILABILITY: CheShift-2 is free of charge for academic use and can be accessed from www.cheshift.com

Based on the results of our recent work on the determination of the potentials of mean force of pairs of models of amino-acid side chains in water, in this work we make an attempt at introducing temperature-dependent side chain - side chain interaction potentials in our coarse-grained UNRES energy function. For hydrophobic pairs as well as oppositely-charged pairs, two functional forms are introduced, one of which implies a linear dependence of the free energy of interactions on temperature and the other one a hyperbolic-tangent dependence. The free energy of the interactions of other pairs is assumed to be independent of temperature. With the example of the N-terminal part of the B-domain of staphylococcal protein A, we demonstrate that, with this temperature dependence, the radius of gyration and the root-mean-square deviation from the native structure grow less steeply with temperature and the heat-capacity peak is lower than that obtained with temperature-independent side chain - side chain potentials. This demonstrates that ignoring the increase of the strength of hydrophobic interactions with increasing temperature in coarse-grained force fields is likely to result in grossly wrong predictions of the thermodynamics of folding and of the process of thermal unfolding made with such force fields.

United-residue models of polypeptide chains [3, 5, 19-22, 24, 31, 33] have long been of interest, because they enable one to carry out global conformational searches of proteins in real time, which in turn can facilitate ab initio protein structure predictions based solely on Anfinsen's thermodynamic hypothesis [1], according to which the native structure of a protein is a global minimum of its potential energy surface [32]. In the last few years, we developed a united-residue force field [20-22, 24], hereafter referred to as UNRES, in which a polypeptide chain is represented by a sequence of α-carbon (Cα) atoms linked by virtual bonds with attached united side chains (SC) and united peptide groups (p) located in the middle between the consecutive α-carbons (Figure 1). Only the united peptide groups and united side chains serve as interaction sites, the α-carbons serving to define the geometry.

A statistical mechanical approach to the protein folding problem is developed based on computer simulations. The properties of proteins related to conformation and folding are determined from the density of states of the protein. A new simulation procedure, the Entropy Sampling Monte Carlo method, is used to determine accurately the density of states of the protein. To enhance the efficiency of sampling the conformational space of a protein, two techniques (a conformational-biased chain regrowth procedure and a jump-walking method) were introduced into the simulation. Applications of the approach to study a number of model polypeptides and a small protein, Bovine Pancreatic Trypsin Inhibitor, have been carried out. The results obtained demonstrate that the new approach is more powerful and produces richer information about the thermodynamics and folding behavior of proteins than conventional simulation methods.

Computer programs that can be used for the design of synthetic genes and that are run on an Apple Macintosh computer are described. These programs determine nucleic acid sequences encoding amino acid sequences. They select DNA sequences based on codon usage as specified by the user, and determine the placement of base changes that can be used to create restriction enzyme sites without altering the amino acid sequence. A new algorithm for finding restriction sites by translating the restriction endonuclease target sequence in all three reading frames and then searching the given peptide or protein amino acid sequence with these short restriction enzyme peptide sequences is described. Examples are given for the creation of synthetic DNA sequences for the bovine prethrombin-2 and ribonuclease A genes.